Article
Reference
Genome-wide copy number variation in epilepsy: novel susceptibility loci in idiopathic generalized and focal epilepsies
MEFFORD, Heather C., et al.
Abstract
Epilepsy is one of the most common neurological disorders in humans with a prevalence of 1% and a lifetime incidence of 3%. Several genes have been identified in rare autosomal dominant and severe sporadic forms of epilepsy, but the genetic cause is unknown in the vast majority of cases. Copy number variants (CNVs) are known to play an important role in the genetic etiology of many neurodevelopmental disorders, including intellectual disability (ID), autism, and schizophrenia. Genome-wide studies of copy number variation in epilepsy have not been performed. We have applied whole-genome oligonucleotide array comparative genomic hybridization to a cohort of 517 individuals with various idiopathic, non-lesional epilepsies. We detected one or more rare genic CNVs in 8.9% of affected individuals that are not present in 2,493 controls; five individuals had two rare CNVs. We identified CNVs in genes previously implicated in other neurodevelopmental disorders, including two deletions in AUTS2 and one deletion in CNTNAP2. Therefore, our findings indicate that rare CNVs are likely to contribute to a broad range of generalized and [...]
MEFFORD, Heather C., et al . Genome-wide copy number variation in epilepsy: novel
susceptibility loci in idiopathic generalized and focal epilepsies. PLOS Genetics , 2010, vol. 6, no. 5, p. e1000962
DOI : 10.1371/journal.pgen.1000962 PMID : 20502679
Available at:
http://archive-ouverte.unige.ch/unige:20651
Disclaimer: layout of this document may differ from the published version.
Susceptibility Loci in Idiopathic Generalized and Focal Epilepsies
Heather C. Mefford1,2*, Hiltrud Muhle3, Philipp Ostertag3, Sarah von Spiczak3, Karen Buysse4, Carl Baker2, Andre Franke5, Alain Malafosse6, Pierre Genton7, Pierre Thomas8, Christina A. Gurnett9, Stefan Schreiber5, Alexander G. Bassuk10, Michel Guipponi6, Ulrich Stephani3, Ingo Helbig3, Evan E. Eichler2,11
1Department of Pediatrics, University of Washington, Seattle, Washington, United States of America,2Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America,3Department of Neuropediatrics, Christian-Albrechts University of Kiel and University Medical Center Schleswig-Holstein, Kiel, Germany,4Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium,5Institute of Clinical Molecular Biology, Christian-Albrechts University, Kiel, Germany,6Department of Genetic Medicine and Development, University of Geneva Medical School and University Hospitals of Geneva, Geneva, Switzerland,7Centre Saint Paul-Hoˆpital Henri Gastaut, Marseilles, France,8Unite´ Fonctionnelle EEG-Epileptologie and Service de Neurologie, Hoˆpital Pasteur, Nice, France,9Department of Neurology, Washington University, St. Louis, Missouri, United States of America,10Department of Pediatrics, University of Iowa, Iowa City, Iowa, United States of America, 11Howard Hughes Medical Institute, University of Washington, Seattle, Washington, United States of America
Abstract
Epilepsy is one of the most common neurological disorders in humans with a prevalence of 1% and a lifetime incidence of 3%. Several genes have been identified in rare autosomal dominant and severe sporadic forms of epilepsy, but the genetic cause is unknown in the vast majority of cases. Copy number variants (CNVs) are known to play an important role in the genetic etiology of many neurodevelopmental disorders, including intellectual disability (ID), autism, and schizophrenia.
Genome-wide studies of copy number variation in epilepsy have not been performed. We have applied whole-genome oligonucleotide array comparative genomic hybridization to a cohort of 517 individuals with various idiopathic, non-lesional epilepsies. We detected one or more rare genic CNVs in 8.9% of affected individuals that are not present in 2,493 controls;
five individuals had two rare CNVs. We identified CNVs in genes previously implicated in other neurodevelopmental disorders, including two deletions inAUTS2and one deletion inCNTNAP2. Therefore, our findings indicate that rare CNVs are likely to contribute to a broad range of generalized and focal epilepsies. In addition, we find that 2.9% of patients carry deletions at 15q11.2, 15q13.3, or 16p13.11, genomic hotspots previously associated with ID, autism, or schizophrenia. In summary, our findings suggest common etiological factors for seemingly diverse diseases such as ID, autism, schizophrenia, and epilepsy.
Citation:Mefford HC, Muhle H, Ostertag P, von Spiczak S, Buysse K, et al. (2010) Genome-Wide Copy Number Variation in Epilepsy: Novel Susceptibility Loci in Idiopathic Generalized and Focal Epilepsies. PLoS Genet 6(5): e1000962. doi:10.1371/journal.pgen.1000962
Editor:Wayne N. Frankel, The Jackson Laboratory, United States of America ReceivedJanuary 28, 2010;AcceptedApril 20, 2010;PublishedMay 20, 2010
Copyright:ß2010 Mefford et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:This work is supported, in part, by U.S. National Institutes of Health grants HD043569 to EEE, HD043376 to HCM, and NS064159 to AGB. HCM holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund. EEE is an investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests:The authors have declared that no competing interests exist.
* E-mail: hmefford@u.washington.edu
Introduction
Epilepsy is one of the most common neurological disorders in humans with a prevalence of,1% and a lifetime incidence of up to 3% [1]. The epilepsies present with a broad range of clinical features, and over 50 distinct epilepsy syndromes are now recognized.
Particularly in a pediatric setting, a broad range of different epilepsy syndromes can be distinguished. Seizure disorders can roughly be divided into idiopathic or symptomatic epilepsies. While symptomatic epilepsies are due to an identifiable cause such as metabolic disorders, brain trauma or intracranial tumors, idiopathic seizure disorders occur in the absence of identifiable causal factors and are thought to have a strong genetic contribution.
Although it has long been observed that the idiopathic epilepsies have a genetic component, the genetic etiology of only a small fraction of cases can be determined. The role of copy number
variants (CNVs) in intellectual disability (ID) [2–8], autism [9–14]
and schizophrenia [15–19] has been extensively investigated. It has become increasingly clear that, collectively, rare variants contribute significantly to the etiology of these common diseases–following the rare variant common disease hypothesis. We hypothesize this can be extended to other neurological disorders and that rare CNVs significantly contribute to the genetic etiology of epilepsy.
Recently, in a study targeted to six genomic regions, recurrent microdeletions on chromosome 15q13.3, 16p13.11 and 15q11.2 were identified as important genetic factors predisposing to idiopathic generalized epilepsy (IGE) [20–22]. Here, we carry out whole-genome array comparative genomic hybridization (CGH) in a cohort of 517 individuals with mixed types of idiopathic epilepsy in order to discover novel copy number changes associated with epilepsy. We find recurrent microdeletions of 15q13.3, 16p13.11 and 15q11.2 each in ,1% of affected
individuals, confirming previous studies [20–22]. In addition to recurrent rearrangements at rearrangement-prone regions, we show that, overall, 8.9% of affected individuals have one or more rare copy number changes involving at least one gene.
Results
We performed genome-wide array CGH to detect copy number changes in 517 patients with mixed types of epilepsy. Of these, 399
have idiopathic generalized epilepsy (IGE), 50 have benign epilepsy with centrotemporal spikes (BECTS) and 68 have other types of idiopathic seizure disorders (Table 1). We used a custom microarray with high-density targeted coverage of 107 regions of the genome flanked by large, highly homologous duplications, termed rearrangement hotspots [23]. In addition, probes were evenly spaced throughout the remainder of the genome with average probe spacing of ,38 kb. Overall, we find that 46 probands (8.9%) carry one or more rare CNVs not previously reported in the 2493 unrelated controls [24]. The rare CNVs detected in our cohort range in size from 13 kb to 15.9 Mb (average 1.2 Mb; median 600 kb), and the majority (69%) are deletion events.
Rearrangements at genomic hotspots
We first evaluated rearrangement hotspots for copy number changes. We found 20 probands (3.9%) with copy number changes at known rearrangement hotspots including 15q13.3 deletions (n = 5), 16p13.11 deletions (n = 5), 15q11.2 BP1–BP2 deletions (n = 5), 1q21.1 deletions (n = 2), a 16p12.1 deletion (n = 1), a 16p11.2 duplication (n = 1) and a more distal 16p11.2 deletion (n = 1) (Table 2, Figure 1). We also identified four individuals with duplications of 15q11.2 BP1–BP2; because duplications of this region are frequent in the general population, we classified these duplications as polymorphic events. These results confirm our previous studies and emphasize the importance of deletions of 15q13.3, 16p13.11 and 15q11.2 BP1–BP2 as frequent genetic susceptibility factors in epilepsy [20–22]. All three regions have also been associated with ID, autism and/or schizophrenia [15,17,25–32], as have deletions at 1q21.1 [33,34], two distinct regions of 16p11.2 [10,14,35–37] and Author Summary
Epilepsy, a common neurological disorder characterized by recurrent seizures, affects up to 3% of the population. In some cases, the epilepsy has a clear cause such as an abnormality in the brain or a head injury. However, in many cases there is no obvious cause. Numerous studies have shown that genetic factors are important in these types of epilepsy, but although several epilepsy genes are known, we can still only identify the genetic cause in a very small fraction of cases. In order to identify new genes that contribute to the genetic causes of epilepsy, we searched the human genome for deletions (missing copies) and duplications (extra copies) of genes in,500 patients with epilepsy that are not found in control individuals. Using this approach, we identified several large deletions that are important in at least 3% of epilepsy cases. Furthermore, we found new candidate genes, some of which are also thought to play a role in other related disorders such as autism and intellectual disability. These genes are candi- dates for further studies in patients with epilepsy.
Table 1.Phenotypes of probands evaluated by array CGH.
Type of epilepsy N
Hotspot CNVs detected
Other CNVs
detected Total
IGE (n = 399)
Juvenile myoclonic epilepsy (JME) 189 8‘ 9‘ 17
Absence epilepsy (AE) 94 5 5* 10
IGE with GTCS only 33 0 2 2
IGE unclassified 63 2 4 6
Benign myoclonic epilepsy of infancy 5 0 0 0
Myoclonic astatic epilepsy (MAE) 15 0 2 2
Idiopathic focal epilepsy (n = 63)
BECTS 50 3 2 5
ABPE 13 0 0 0
Other (n = 55)
ESES 4 0 0 0
Landau-Kleffner syndrome 3 0 0 0
Severe IGE of infancy (SIGEI) 15 1 1 2
West syndrome 4 0 2* 2
IC/NC 10 1 2* 3
Unclassified 19 0 2 2
Total 517 20 31 51
IGE, idiopathic generalized epilepsy; GTCS, generalized tonic-clonic seizures; BECTS, benign epilepsy with centrotemporal spikes; ABPE, atypical benign partial epilepsy;
ESES, electrical status epilepticus during slow-wave sleep; IC, infantile convulsions; NC, neonatal convulsions;
*indicates two events in a single individual;
‘two individuals (EMJ071 and EMJ117) each carrying one hotspot and one non-hotspot event.
doi:10.1371/journal.pgen.1000962.t001
CNVs in Epilepsy
Table 2.Rare copy number variants in 517 patients with epilepsy.
Case
Chromosome
Location HS
Coordinates
(build36; Mb) Size CNV Inheritance Phenotype
RefSeq genes (n)
Possible candidate genes Idopathic Generalized Epilepsies (n = 399)
EMJ 049 1q21.1 Y Chr1: 145.0–145.9 900 kb Del - JME 8 GJA8
ND02006 15q11.2 Y Chr15: 20.2–20.8 600 kb Del - IAE 4 CYFIP1
ND03383 15q11.2 Y Chr15: 20.2–20.8 600 kb Del - CAE 4 CYFIP1
ND06631 15q11.2 Y Chr15: 20.2–20.8 600 kb Del Inh (P) CAE 4 CYFIP1
K004 15q11.2 Y Chr15: 20.2–20.8 600 kb Del Inh (P) IGE 4 CYFIP1
EPI 62 15q13.3 Y Chr15: 28.7–30.1 1.4 Mb Del - IAE 6 CHRNA7
EMJ 001** 15q13.3 Y Chr15: 28.7–30.1 1.4 Mb Del - JME 6 CHRNA7
EMJ 002 15q13.3 Y Chr15: 28.7–30.1 1.4 Mb Del - JME 6 CHRNA7
EMJ 020** 15q13.3 Y Chr15: 28.7–30.1 1.4 Mb Del - JME 6 CHRNA7
IA G5 15q13.3 Y Chr15: 28.7–30.1 1.4 Mb Del - IGE+ID 6 CHRNA7
EMJ 162 16p11.2 Y Chr16: 29.5–30.2 700 kb Dup - JME 30 SEZ6L2
ND3074 16p13.11 Y Chr16:15.4–16.3 900 kb Del Inh (M) CAE 6 NDE1
EPI 17 16p13.11 Y Chr16:15.4–16.3 900 kb Del - JME 6 NDE1
EMJ 071* 16p13.11 Y Chr16:15.4–16.3 900 kb Del - JME 6 NDE1
EMJ 117* 16p13.11 Y Chr16: 15.4–18.5 3.1 Mb Del - JME 7 NDE1
ND05586 1p31.1 Chr1: 72.04–72.15 111.3 kb Del Inh (P) CAE 1 NEGR1
ND05260* 4q22.2 Chr4: 94.18–94.83 646.6 kb Del Inh (M) CAE 1 GRID2
K 111 5p15.33 Chr5: 0.72–1.43 713.0 kb Dup Inh (M‘) MAE 10 NKD2, SLCA18
EP007.1 5q33.2 Chr5: 153.2–160.3 7.1 Mb Del Not in M IGE+ID 44 CYFIP2
EMJ 005 6q12 Chr6: 65.03–66.09 1.06 Mb Dup - JME 1 EYS
ND01440 7q11.22 Chr7: 69.38–69.46 78.7 kb Del - JME 1 AUTS2
K 039 7q36.1 Chr7: 151.35–151.43 85.8 kb Del Inh (P) MAE 1 GALNT11
ND03578 8q21-q22 Chr8: 83.97–97.20 15.9 Mb Dup Inh (P) JME+ID 50 many
EMJ 013 9p21.3 Chr9: 21.21–21.63 427.5 kb Del - JME 9 KLHL9
EP005.1 9q21.32 Chr9: 83.9–85.2 1.30 Mb Del Inh (M) IGE 2 RASEF
ND05260* 9q31.3 Chr9: 113.33–114.33 1.01 Mb Dup Inh (M) CAE 10
EMJ 071* 13q31.1 Chr13: 84.69–85.36 671.8 kb Del - JME 1 SLITRK6
EMJ 067 14q24.2 Chr14: 70.96–71.23 268.6 kb Del - JME 1 SIPA1L1
EPI 66 15q25.2 Chr15: 83.00–83.12 117.4 kb Dup - IAE 3 NBM
ND03244 16q23.1 Chr16: 74.49–75.27 785.8 kb Dup - GTCS only 1 CNTNAP4
EPI 52 17p11.2 Chr17: 19.92–19.94 13.3 kb Del - GTCS only 1 CYTSB
EMJ 117* 17p11.2 Chr17: 19.92–19.94 17.5 kb Del - JME 1 CYTSB
EPI 40 17q12 Chr17: 30.53–30.87 338.5 kb Del - IAE 7 UNC45
EMJ 039 18q11.2 Chr18: 19.66–20.50 840.4 kb Dup - JME 6
EMJ 069 18q11.2 Chr18: 19.66–20.50 840.4 kb Dup - JME 6
ND02416 21q21.1 Chr21: 16.21–18.81 2.59 Mb Dup Inh (M) IGE+ID 7
EPI 26 Xp22.31 ChrX: 7.78–8.39 605.5 kb Dup - IGE 4 PNPLA4
Idiopathic Focal Epilepsies (n = 63)
EPI 60 1q21.1 Y Chr1: 145.0–145.9 900 kb Del - BECTS 8
K 105 16p12.1 Y Chr16: 21.8–22.3 500 kb Del - BECTS 7
EPI 21 16p13.11 Y Chr16: 15.4–16.3 900 kb Del - BECTS 6 NDE1
EPI 58 4q35.1 Chr4: 186.30–186.61 302.4 kb Dup - BECTS 8 SLC25A, SNX25
K 093 8p23.1 Chr8: 10.19–10.37 173.1 kb Del - BECTS 1 MSRA
Other (n = 55)
K 047 15q11.2 Y Chr15: 20.2–20.8 600 kb Del brother‘ IC 4 CYFIP1
K 027 16p11.2 Y Chr16:28.7–28.9 200 kb Del - SIGEI 9
K 109 2q35 Chr2: 218.36–218.94 571.9 kb Dup - SIGEI 11
16p12 [38], which were also detected in our cohort. Deletions of 16p13.11 (5/517 vs 0/2493 controls, p = 0.00014, Fisher’s exact test), 15q13.3 (5/517 vs 0/2493, p = 0.00014) and 15q11.2 (5/517 vs. 4/2493, p = 0.010) are significantly enriched in our epilepsy cohort and together account for 2.9% of cases.
Rare or unique deletions involving potential candidate genes
We next focused on non-hotspot CNVs that overlap one or more genes and are not present in the control cohort of 2493 individuals [24]. We identified 28 individuals with at least one rare gene-containing deletion or duplication, and five individuals each carry two rare CNVs (Table 2). Fifteen of the events we detected involve a single gene. Two genes were altered in two patients each:
AUTS2 deletions were identified in one proband with juvenile myoclonic epilepsy (JME) and one proband with unclassified non- lesional epilepsy with features of atypical benign partial epilepsy (ABPE) [39]. Deletions involvingCTYSB(SPECC1) were identified in two probands with IGE. All other single-gene CNVs were seen only once. Seventeen events involved multiple genes, one of which was observed in two different individuals with JME (duplication of 18q11, Table 2).
Individuals with multiple rare CNVs
We found five individuals with two rare CNVs (Figure 2). Two patients with JME and a deletion of 16p13.11 (EMJ071 and EMJ117) each have a second rare deletion. EMJ071 has a large deletion on chromosome 13 that removes the SLITRK6gene, a member of the SLITRK gene family involved in controlling neurite outgrowth; individual EMJ117 also has a deletion involving the CTYSB gene. Case ND05260 (childhood absence epilepsy, CAE) carries a 647-kb deletion within theGRID2gene, which encodes a glutamate receptor expressed in the cerebellum, and a 1-Mb duplication of 9q31. Though both are maternally inherited, neither has been reported in controls. Case EPI 51 (idiopathic West syndrome) has two apparently independent duplications of chromosome 5q35, each containing several genes.
Finally, we identified one proband with neonatal convulsions (NC) carrying a deletion within theCNTNAP2gene that spans exons 2–
4 as well as a 370-kb deletion of 17p13 involving 7 genes.
DNA from one of more family members was available for analysis in 14 cases. Inheritance, if determined, is shown in
Table 2. In twelve cases, we determined that one or both CNVs in the proband were inherited; in three cases the transmitting parent is also affected. In one case (EP007.1), the CNV was not found in the mother, but the father was unavailable. In another case (K047), parents were unavailable, but a brother was found to carry the same CNV suggesting one of the parents carries the same CNV.
Discussion
In this study, we performed whole-genome array CGH in a series of 517 individuals with a presenting diagnosis of idiopathic epilepsy in order to discover novel copy number changes associated with epilepsy. While our previous studies were targeted to specific genomic regions in probands with IGE [21,22], here we present data from whole-genome analysis on probands with IGE and extend our analysis to other idiopathic epilepsy syndromes. In total, we identified 46 individuals (8.9%) with 51 rearrangements that may be pathogenic as they were not found in controls or were significantly enriched in our epilepsy cohort.
Hotspot rearrangements
Rearrangements at several genomic hotspots have been associated with a range of neurocognitive disorders. In our cohort of 517 probands with epilepsy, we find deletions at 15q13.3, 16p13.11 and 15q11.2 in 2.9% of our cases. Interestingly, all of the deletions of 15q13.3 (n = 5) and 4/5 deletions at 16p13.11 and 15q11.2 were in probands with IGE, accounting for 3.3% of the patients with IGE in our cohort confirming our previous findings.
While it is possible that deletions of 15q13.3 are also predisposing to non-IGE epilepsy syndromes, we did not find this to be the case in our series (n = 118). Additional large cohorts of patients with focal epilepsy or epileptic encephalopathy will be required to determine whether these deletions also play a significant role in other subtypes of epilepsy.
Deletions of 16p13.11 have previously been associated with intellectual disability+/2congenital anomalies in one study [26].
Three of four probands with 16p13.11 deletions in that series had epilepsy; two further fetal cases had brain abnormalities. The findings in this cohort and one previous study of IGE [20] suggest that deletions of 16p13.11 are more frequent in epilepsy (0.5–1%
of cases) than in other phenotypes including ID and autism [26,27,32], and may be as frequent as 15q13.3 deletions in Table 2.Cont.
Case
Chromosome
Location HS
Coordinates
(build36; Mb) Size CNV Inheritance Phenotype
RefSeq genes (n)
Possible candidate genes
EPI 51* 5q35.1 Chr5: 167.62–167.89 268.7 kb Dup - West 4 WWC1
EPI 51* 5q35.1 Chr5: 169.43–169.64 230.0 kb Dup - West 4 DOCK2, FOXI1
K 054 7q11.22 Chr7: 69.38–69.42 38.3 kb Del - Unclassified 1 AUTS2
K034* 7q35 Chr7:146.06–146.36 304.4 kb Del Inh (P‘) NC 1 CNTNAP2
ND08273 15q13.3-q14 Chr15: 30.66–32.44 1.78 Mb Dup Inh (M) Unclassified 15
K034* 17p13.1 Chr17:10.36–10.72 370 kb Del Inh (P‘) NC 7 MYH1-3; SCO
HS, hotspot region; Del, deletion; Dup, duplication; Inh, inherited; M, maternal; P, paternal;
‘affected; -, parents unavailable; JME, juvenile myoclonic epilepsy; IAE, idiopathic absence epilepsy; CAE childhood absence epilepsy; IGE, idiopathic generalized epilepsy; GTCS, generalized tonic clonic seizures only; ID, intellectual disability; BECTS, benign epilepsy with centrotemporal spikes; IC, infantile convulsions; SIGEI, several idiopathic generalized epilepsy of infancy; NC, neonatal convulsions;
*two CNVs detected in same individual;
**15q13 deletions previously detected by MLPA [60].
doi:10.1371/journal.pgen.1000962.t002
CNVs in Epilepsy
individuals with IGE. Deletions and duplications of this region have also been reported in schizophrenia, though the associations have not been statistically significant [16,29].
Deletions of 15q13.3, detected in five individuals with IGE in our series, have been associated with a wide range of phenotypes including ID, autism, epilepsy and schizophrenia [15,17,20–
22,25,28,30,31,40]. The gene within the 15q13.3 region that is most likely responsible for the epilepsy phenotype isCHRNA7, a subunit of the nicotinic acetylcholine receptor. At least two small studies have failed to identify causal point mutations in the CHRNA7 gene in autosomal dominant nocturnal frontal lobe epilepsy [41] and JME [42], but additional studies should be performed to further evaluate affected individuals for mutations.
A recent publication identifying atypical rearrangements with exclusive deletions of CHRNA7 further emphasizes the impor- tance of CHRNA7 as the main candidate gene in this region [43].
Compared to the above structural genomic variants, copy number variation at 15q11.2 between breakpoints BP1 and BP2 of the Prader-Willi and Angelman syndrome region is more common in the general population with the BP1–BP2 deletion present in 0.2% of unaffected individuals. Despite this, deletions between BP1 and BP2 have now been reported as enriched in patients with schizophrenia [16,17], ID [27] and epilepsy [20]. Furthermore, there is evidence that patients with Prader-Willi or Angelman syndrome who have deletions including BP1–BP2 are more severely affected [44–46]. In this study, we also find enrichment of deletions at this locus in affected individuals. Together, these studies suggest that deletion of the 15q11.2 BP1–BP2 region confers susceptibility to a wide range of neuropsychiatric conditions, albeit with incomplete penetrance.
Two patients in our series, one each with JME and BECTS, have deletions of 1q21.1, which have been previously associated with a wide range of phenotypes, including intellectually disability and developmental delay [33,34], schizophrenia [15,17,18], congenital heart disease [47,48] and cataracts [34,49]. In two large studies of patients who present primarily with cognitive or developmental delay, 5/42 (11.9%) patients also had seizures [33,34]; 1 of 10 patients with schizophrenia and a 1q21.1 deletion also had epilepsy [15]. Identifying 1q21.1 microdeletions in patients with idiopathic generalized and idiopathic focal epilepsies suggests that variation at this locus predisposes to a broad range of seizure disorders crossing traditional diagnostic boundaries.
In addition, we identified one patient (EMJ162) with JME and a duplication of 16p11.2 (chr16: 29.5–30.2 Mb), which has been associated with autism, developmental delay and schizophrenia [10–12,14,27,35,37]. Finally, we identified one individual with severe idiopathic generalized epilepsy of infancy (SIGEI) (K027) with a more distal deletion of 16p11.2 (chr16: 27.7–28.9 Mb), recently associated with severe early-onset obesity and ID [36], and one patient with BECTS (K105) and a deletion of 16p12.1 (chr16: 20.2–20.8 Mb), also associated with ID and other neurodevelopmental defects [38]. Thus, our data adds to the phenotypic spectrum associated with rearrangements at several genomic hotspot regions. In particular, we identify hotspot deletions in two patients with BECTS. Gene identification in
BECTS, despite representing the most common focal epilepsy syndrome of childhood, has been elusive so far. Here, we suggest that some recurrent hotspot deletions might predispose to both idiopathic generalized and focal epilepsies.
Non-hotspot rearrangements
We detected 18 deletions and 16 duplications that are not associated with rearrangement hotspots. Fifteen events involve a single gene; of these, 12 are deletions. Although all of the CNVs reported here are not found in our control set of 2493 individuals, it is possible that some are rare but benign CNVs. However, many of the CNVs we identified contain one of more plausible candidate genes for epilepsy (Table 2).
We identified a deletion of exons 2–4 in theCNTNAP2gene in a proband with neonatal seizures.CNTNAP2has been identified as a candidate gene for autism [50–52], and heterozygous deletions involving the gene were reported in three patients with schizophrenia and autism [53]. The deletion is predicted to cause an in-frame deletion of 153 amino acids in the resulting protein.
The same patient has a 370-kb deletion of 17p13 that deletes seven genes and has not been seen in our control cohort. We also identified a patient with a duplication encompassing a related gene, CNTNAP4. Finally, two individuals in our cohort have overlapping deletions withinAUTS2. This gene is disrupted byde novo balanced translocations in three unrelated individuals with mental retardation [54] and a pair of twins with autism and mental retardation [55], suggesting a role forAUTS2in normal cognitive development. The two deletions we detected are intragenic and overlapping.
CNVs in epilepsy subtypes
Previous studies of CNVs in epilepsy have focused on probands with IGE. It is known from studies of families with autosomal dominant epilepsy that a wide range of seizure types can be caused by the same single-gene mutation. For example, Dravet syndrome, a severe early-onset disorder associated with poor cognitive outcome, and the milder generalized epilepsy with febrile seizures plus (GEFS+) syndrome are both caused by mutations in the SCN1A gene [56–58]. Therefore, we included probands with common idiopathic focal epilepsies and non-lesional, idiopathic epilepsies. Some of our probands were diagnosed with specific epilepsy syndromes, including myoclonic astatic epilepsy (Doose Syndrome), atypical benign partial epilepsy [39], Landau-Kleffner syndrome, idiopathic West syndrome, severe idiopathic general- ized epilepsy of infancy [59] and benign neonatal or infantile seizures. These particular epilepsy syndromes are usually associ- ated with normal MRI results. We find that 6.6% of probands with IGE and 7.9% of those with idiopathic focal epilepsy harbor rare CNVs that may underlie their epilepsy phenotype. Notably, 12.7%
of patients with other, often more severe forms of epilepsy in our series carry one or more rare CNVs. In our series, the vast majority of patients with deletions of 15q13.3, 16p13.11 and 15q11.2 BP1–BP2 were in the IGE cohort, accounting for 3.3% of cases. In the non-IGE patients, a deletion of 15q11.2 was found in a single patient with infantile seizures and a deletion of 16p13.11 was found in one patient with BECTS, suggesting that deletions at Figure 1. Deletions and duplications at genomic rearrangement hotspots in 20 probands.Array CGH results are depicted for (A) 15q13.3, chr15: 28.0–31.0 Mb, (B) 16p13.11, chr16: 14.5–18.5 Mb, (C) 15q11.2, chr15: 20.0–20.9 Mb, (D) 1q21.1, chr1: 144.0–147.5 Mb, (E) 16p12.1, chr16: 21.6–
22.6 Mb, (F) 16p11.2, chr16:28.6–29.1 Mb, and (G) 16p11.2, chr16: 29.0–30.3 Mb. For each individual, deviations of probe log2ratios from 0 are depicted by gray and black lines. Those exceeding a threshold of 1.5 s.d. from the mean probe ratio are colored green and red to represent relative gains and losses, respectively. Segmental duplications of increasing similarity (90–98%, 98–99%, and.99%) are represented by gray, yellow, and orange bars, respectively. RefSeq genes are depicted in blue.
doi:10.1371/journal.pgen.1000962.g001
CNVs in Epilepsy
these three genomic hotspots confer greater risk for IGE than other types of epilepsy.
In summary, we find that 46/517 probands (8.9%) with various forms of idiopathic epilepsy carry one or more rare CNVs that may predispose to seizures, a frequency similar to that in studies of patients who present with other neurocognitive phenotypes, including ID, autism and schizophrenia. Furthermore, we identified CNVs involving genes and genomic regions previously identified in patients with the neurocognitive phenotypes listed above, suggesting common genetic etiological factors for these disorders. Our data suggest that rare CNVs are important in many subtypes of idiopathic epilepsies, including idiopathic generalized and idiopathic focal epilepsies as well as specific idiopathic, non-lesional epilepsy syndromes. The genomic regions and genes identified in this study are potential novel candidate genes for epilepsy.
Materials and Methods Ethics statement
Patients were collected at five centers after appropriate human subjects approval and informed consent at each site.
Patient cohorts
Patients were collected at five centers: (1) 140 probands with a primary diagnosis of JME, CAE, absence epilepsy, IGE or idiopathic epilepsy were selected from the NINDS repository (http://ccr.coriell.
org/ninds); (2) 160 patients are probands with a primary diagnosis of JME from Switzerland. Patients from cohorts (1) and (2) were previously analyzed using MLPA for theCHRNA7gene [60], and two probands (EMJ001 and EMJ020) were determined to have 15q13.3 microdeletions by that method; they were not previously analyzed for any other copy number changes. (3) 186 German patients came from two cohorts: 76 patients from a population-based cohort from Northern Germany (POPGEN cohort) and 110 patients with childhood-onset epilepsy collected at the University of Kiel. Finally, 41 patients with various idiopathic generalized epilepsies collected at (4) the University of Iowa and (5) at Washington University, St. Louis.
DNA from the NINDS repository was derived from cell lines; DNA from all other cohorts was directly from blood. Patients were diagnosed according to the widely used 1989 ILAE classification [61].
In addition, several pediatric patients were diagnosed with specific syndromes not yet recognized in the ILAE classification (Table 1).
Patients with non-lesional, idiopathic epilepsies in which diagnostic criteria of the recent ILAE classification for particular epilepsy syndromes were not met were labeled as ‘‘unclassified’’.
Array comparative genomic hybridization (CGH)
Array CGH was performed using either custom or commer- cially available oligonucleotide arrays containing 135,000 isother-
mal probes (Roche NimbleGen, Inc.). Customized arrays (459 samples) were designed with higher density probe coverage in known rearrangement hotspot regions (average probe spacing 2.5 kb) with lower density whole-genome backbone coverage (average probe spacing 38 kb). A subset of samples (n = 62) was analyzed using a commercially available whole-genome array (Roche NimbleGen 126135 k whole-genome tiling array) with average probe spacing throughout the genome of 21 kb.
Data analysis
Data were analyzed according to manufacturer’s instructions using NimbleScan software to generate normalized log2 fluorescence intensity ratios. Then, for each sample, normalized log intensity ratios are transformed into z-scores using the chromosome-specific mean and standard deviation. Z-scores are subsequently used to classify probes as ‘‘increased’’, ‘‘normal’’ and ‘‘decreased’’ copy- number using a three-state Hidden Markov Model (HMM). The HMM was implemented using HMMSeg [62], which assumes Gaussian emission probabilities. The ‘‘increased’’ and ‘‘decreased’’
states are defined to have the same standard deviation as the
‘‘normal’’ state but with mean z-score two standard deviations above and below the mean, respectively. Probe-by-probe HMM state assignments are merged into segments according to the following criteria: consecutive probes of the same state less than 50 kb apart are merged, and if two segments of the same state are separated by an intervening sequence of#5 probes and#10 kb, both segments and intervening sequence are called as a single variant. CNV calls are filtered to eliminate (i) events containing,5 probes, (ii) CNVs with .50% overlap in a series of 2493 control individuals [24] and (iii) events that had no overlap with RefSeq genes. In addition, when comparing CNV calls to control CNVs, we eliminated calls for which there was insufficient probe coverage (,5 probes) in the control data to identify the same or similar CNV. Filtered copy number changes are also visually inspected in a genome browser.
Acknowledgments
This study used samples from the NINDS Human Genetics Resource Center DNA and Cell Line Repository (http://ccr.coriell.org/ninds), as well as clinical data. NINDS Repository sample numbers corresponding to the samples used are available upon request.
Author Contributions
Conceived and designed the experiments: HCM US IH EEE. Performed the experiments: HCM KB CB. Analyzed the data: HCM CB IH.
Contributed reagents/materials/analysis tools: HCM HM PO SvS AF AM PG PT CAG SS AGB MG US IH EEE. Wrote the paper: HCM. Provided clinical data: HM PO SvS.
References
1. Hauser WA, Annegers JF, Rocca WA (1996) Descriptive epidemiology of epilepsy: contributions of population-based studies from Rochester, Minnesota.
Mayo Clin Proc 71: 576–586.
2. de Vries BB, Pfundt R, Leisink M, Koolen DA, Vissers LE, et al. (2005) Diagnostic genome profiling in mental retardation. Am J Hum Genet 77: 606–616.
3. Friedman JM, Baross A, Delaney AD, Ally A, Arbour L, et al. (2006) Oligonucleotide microarray analysis of genomic imbalance in children with mental retardation. Am J Hum Genet 79: 500–513.
4. Koolen DA, Vissers LE, Pfundt R, de Leeuw N, Knight SJ, et al. (2006) A new chromosome 17q21.31 microdeletion syndrome associated with a common inversion polymorphism. Nat Genet 38: 999–1001.
5. Sagoo G, Butterworth A, Sanderson S, Shaw-Smith C, Higgins J, et al. (2009) Array CGH in patients with learning disability (mental retardation) and congenital anomalies: updated systematic review and meta-analysis of 19 studies and 13,926 subjects. Genet Med 11: 139–146.
6. Shaffer LG, Kashork CD, Saleki R, Rorem E, Sundin K, et al. (2006) Targeted genomic microarray analysis for identification of chromo- some abnormalities in 1500 consecutive clinical cases. J Pediatr 149:
98–102.
7. Sharp AJ, Hansen S, Selzer RR, Cheng Z, Regan R, et al. (2006) Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome. Nat Genet 38: 1038–1042.
Figure 2. Two rare CNVs in five probands.Array CGH results are shown for the for two rare CNVs detected in probands EMJ071 (A), ND05260 (B), EPI51 (C), K034 (D), and EMJ117 (E). Array CGH results are depicted as in Figure 1; segmental duplications are not shown in this figure.
doi:10.1371/journal.pgen.1000962.g002
CNVs in Epilepsy
8. Shaw-Smith C, Pittman AM, Willatt L, Martin H, Rickman L, et al. (2006) Microdeletion encompassing MAPT at chromosome 17q21.3 is associated with developmental delay and learning disability. Nat Genet 38: 1032–1037.
9. Christian SL, Brune CW, Sudi J, Kumar RA, Liu S, et al. (2008) Novel submicroscopic chromosomal abnormalities detected in autism spectrum disorder. Biol Psychiatry 63: 1111–1117.
10. Kumar RA, KaraMohamed S, Sudi J, Conrad DF, Brune C, et al. (2008) Recurrent 16p11.2 microdeletions in autism. Hum Mol Genet 17: 628–638.
11. Marshall CR, Noor A, Vincent JB, Lionel AC, Feuk L, et al. (2008) Structural variation of chromosomes in autism spectrum disorder. Am J Hum Genet 82:
477–488.
12. Sebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, et al. (2007) Strong association of de novo copy number mutations with autism. Science 316:
445–449.
13. Szatmari P, Paterson AD, Zwaigenbaum L, Roberts W, Brian J, et al. (2007) Mapping autism risk loci using genetic linkage and chromosomal rearrange- ments. Nat Genet 39: 319–328.
14. Weiss LA, Shen Y, Korn JM, Arking DE, Miller DT, et al. (2008) Association between microdeletion and microduplication at 16p11.2 and autism.
N Engl J Med 358: 667–675.
15. International Schizophrenia Consortium (2008) Rare chromosomal deletions and duplications increase risk of schizophrenia. Nature 455: 237–241.
16. Kirov G, Grozeva D, Norton N, Ivanov D, Mantripragada KK, et al. (2009) Support for the involvement of large cnvs in the pathogenesis of schizophrenia.
Hum Mol Genet 18: 1497–1503.
17. Stefansson H, Rujescu D, Cichon S, Pietilainen OP, Ingason A, et al. (2008) Large recurrent microdeletions associated with schizophrenia. Nature 455:
232–236.
18. Walsh T, McClellan JM, McCarthy SE, Addington AM, Pierce SB, et al. (2008) Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia. Science 320: 539–543.
19. Xu B, Roos JL, Levy S, van Rensburg EJ, Gogos JA, et al. (2008) Strong association of de novo copy number mutations with sporadic schizophrenia. Nat Genet 40: 880–885.
20. de Kovel CG, Trucks H, Helbig I, Mefford HC, Baker C, et al. (2009) Recurrent microdeletions at 15q11.2 and 16p13.11 predispose to idiopathic generalized epilepsies. Brain.
21. Dibbens LM, Mullen S, Helbig I, Mefford HC, Bayly MA, et al. (2009) Familial and sporadic 15q13.3 microdeletions in idiopathic generalized epilepsy:
precedent for disorders with complex inheritance. Hum Mol Genet 18:
3626–3631.
22. Helbig I, Mefford HC, Sharp AJ, Guipponi M, Fichera M, et al. (2009) 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy. Nat Genet 41:
160–162.
23. Bailey JA, Gu Z, Clark RA, Reinert K, Samonte RV, et al. (2002) Recent segmental duplications in the human genome. Science 297: 1003–1007.
24. Itsara A, Cooper GM, Baker C, Girirajan S, Li J, et al. (2009) Population analysis of large copy number variants and hotspots of human genetic disease.
Am J Hum Genet 84: 148–161.
25. Ben-Shachar S, Lanpher B, German JR, Qasaymeh M, Potocki L, et al. (2009) Microdeletion 15q13.3: a locus with incomplete penetrance for autism, mental retardation, and psychiatric disorders. J Med Genet 46: 382–388.
26. Hannes FD, Sharp AJ, Mefford HC, de Ravel T, Ruivenkamp CA, et al. (2009) Recurrent reciprocal deletions and duplications of 16p13.11: the deletion is a risk factor for MR/MCA while the duplication may be a rare benign variant.
J Med Genet 46: 223–232.
27. Mefford HC, Cooper GM, Zerr T, Smith JD, Baker C, et al. (2009) A method for rapid, targeted CNV genotyping identifies rare variants associated with neurocognitive disease. Genome Res 19: 1579–1585.
28. Miller DT, Shen Y, Weiss LA, Korn J, Anselm I, et al. (2008) Microdeletion/
duplication at 15q13.2q13.3 among individuals with features of autism and other neuropsychiatirc disorders. J Med Genet 46: 242–248.
29. Need AC, Ge D, Weale ME, Maia J, Feng S, et al. (2009) A genome-wide investigation of SNPs and CNVs in schizophrenia. PLoS Genet 5: e1000373.
doi:10.1371/journal.pgen.1000373.
30. Pagnamenta AT, Wing K, Akha ES, Knight SJ, Bolte S, et al. (2009) A 15q13.3 microdeletion segregating with autism. Eur J Hum Genet 17: 687–692.
31. Sharp AJ, Mefford HC, Li K, Baker C, Skinner C, et al. (2008) A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures. Nat Genet 40: 322–328.
32. Ullmann R, Turner G, Kirchhoff M, Chen W, Tonge B, et al. (2007) Array CGH identifies reciprocal 16p13.1 duplications and deletions that predispose to autism and/or mental retardation. Hum Mutat 28: 674–682.
33. Brunetti-Pierri N, Berg JS, Scaglia F, Belmont J, Bacino CA, et al. (2008) Recurrent reciprocal 1q21.1 deletions and duplications associated with microcephaly or macrocephaly and developmental and behavioral abnormal- ities. Nat Genet 40: 1466–1471.
34. Mefford HC, Sharp AJ, Baker C, Itsara A, Jiang Z, et al. (2008) Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes.
N Engl J Med 359: 1685–1699.
35. Bijlsma EK, Gijsbers AC, Schuurs-Hoeijmakers JH, van Haeringen A, Fransen van de Putte DE, et al. (2009) Extending the phenotype of recurrent rearrangements of 16p11.2: Deletions in mentally retarded patients without autism and in normal individuals. Eur J Med Genet 52: 77–87.
36. Bochukova EG, Huang N, Keogh J, Henning E, Purmann C, et al. (2010) Large, rare chromosomal deletions associated with severe early-onset obesity. Nature 463: 666–670.
37. McCarthy SE, Makarov V, Kirov G, Addington AM, McClellan J, et al. (2009) Microduplications of 16p11.2 are associated with schizophrenia. Nat Genet 41:
1223–1227.
38. Girirajan S, Rosenfeld JA, Cooper GM, Antonacci F, Siswara P, et al. (2010) A recurrent 16p12.1 microdeletion supports a two-hit model for severe developmental delay. Nat Genet 42: 203–209.
39. Doose H, Hahn A, Neubauer BA, Pistohl J, Stephani U (2001) Atypical
‘‘benign’’ partial epilepsy of childhood or pseudo-lennox syndrome. Part II:
family study. Neuropediatrics 32: 9–13.
40. van Bon BW, Mefford HC, Menten B, Koolen DA, Sharp AJ, et al. (2009) Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome. J Med Genet 46: 511–523.
41. Bonati MT, Combi R, Asselta R, Duga S, Malcovati M, et al. (2002) Exclusion of linkage of nine neuronal nicotinic acetylcholine receptor subunit genes expressed in brain in autosomal dominant nocturnal frontal lobe epilepsy in four unrelated families. J Neurol 249: 967–974.
42. Taske NL, Williamson MP, Makoff A, Bate L, Curtis D, et al. (2002) Evaluation of the positional candidate gene CHRNA7 at the juvenile myoclonic epilepsy locus (EJM2) on chromosome 15q13-14. Epilepsy Res 49: 157–172.
43. Shinawi M, Schaaf CP, Bhatt SS, Xia Z, Patel A, et al. (2009) A small recurrent deletion within 15q13.3 is associated with a range of neurodevelopmental phenotypes. Nat Genet 41: 1269–1271.
44. Butler MG, Bittel DC, Kibiryeva N, Talebizadeh Z, Thompson T (2004) Behavioral differences among subjects with Prader-Willi syndrome and type I or type II deletion and maternal disomy. Pediatrics 113: 565–573.
45. Hartley SL, Maclean WE, Jr., Butler MG, Zarcone J, Thompson T (2005) Maladaptive behaviors and risk factors among the genetic subtypes of Prader- Willi syndrome. Am J Med Genet A 136: 140–145.
46. Sahoo T, Peters SU, Madduri NS, Glaze DG, German JR, et al. (2006) Microarray based comparative genomic hybridization testing in deletion bearing patients with Angelman syndrome: genotype-phenotype correlations. J Med Genet 43: 512–516.
47. Christiansen J, Dyck JD, Elyas BG, Lilley M, Bamforth JS, et al. (2004) Chromosome 1q21.1 contiguous gene deletion is associated with congenital heart disease. Circ Res 94: 1429–1435.
48. Greenway SC, Pereira AC, Lin JC, DePalma SR, Israel SJ, et al. (2009) De novo copy number variants identify new genes and loci in isolated sporadic tetralogy of Fallot. Nat Genet 41: 931–935.
49. Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, et al. (2006) Global variation in copy number in the human genome. Nature 444: 444–454.
50. Alarcon M, Abrahams BS, Stone JL, Duvall JA, Perederiy JV, et al. (2008) Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene. Am J Hum Genet 82: 150–159.
51. Arking DE, Cutler DJ, Brune CW, Teslovich TM, West K, et al. (2008) A common genetic variant in the neurexin superfamily member CNTNAP2 increases familial risk of autism. Am J Hum Genet 82: 160–164.
52. Bakkaloglu B, O’Roak BJ, Louvi A, Gupta AR, Abelson JF, et al. (2008) Molecular cytogenetic analysis and resequencing of contactin associated protein- like 2 in autism spectrum disorders. Am J Hum Genet 82: 165–173.
53. Friedman JI, Vrijenhoek T, Markx S, Janssen IM, van der Vliet WA, et al.
(2008) CNTNAP2 gene dosage variation is associated with schizophrenia and epilepsy. Mol Psychiatry 13: 261–266.
54. Kalscheuer VM, FitzPatrick D, Tommerup N, Bugge M, Niebuhr E, et al.
(2007) Mutations in autism susceptibility candidate 2 (AUTS2) in patients with mental retardation. Hum Genet 121: 501–509.
55. Sultana R, Yu CE, Yu J, Munson J, Chen D, et al. (2002) Identification of a novel gene on chromosome 7q11.2 interrupted by a translocation breakpoint in a pair of autistic twins. Genomics 80: 129–134.
56. Claes L, Ceulemans B, Audenaert D, Smets K, Lofgren A, et al. (2003) De novo SCN1A mutations are a major cause of severe myoclonic epilepsy of infancy.
Hum Mutat 21: 615–621.
57. Escayg A, MacDonald BT, Meisler MH, Baulac S, Huberfeld G, et al. (2000) Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2. Nat Genet 24: 343–345.
58. Fujiwara T (2006) Clinical spectrum of mutations in SCN1A gene: severe myoclonic epilepsy in infancy and related epilepsies. Epilepsy Res 70 Suppl 1:
S223–230.
59. Doose H, Lunau H, Castiglione E, Waltz S (1998) Severe idiopathic generalized epilepsy of infancy with generalized tonic-clonic seizures. Neuropediatrics 29:
229–238.
60. Helbig I, Mefford HC, Sharp AJ, Guipponi M, Fichera M, et al. (2009) 15q13.3 microdeletions increase risk of idiopathic generalized epilepsy. Nat Genet 41:
160–162.
61. ILAE (1989) Proposal for revised classification of epilepsies and epileptic syndromes. Commission on Classification and Terminology of the International League Against Epilepsy. Epilepsia 30: 389–399.
62. Day N, Hemmaplardh A, Thurman RE, Stamatoyannopoulos JA, Noble WS (2007) Unsupervised segmentation of continuous genomic data. Bioinformatics 23: 1424–1426.
